Phage-Mediated Bioluminescent Detection of Yersinia Pestis

ABSTRACT

The present disclosure relates to compositions, methods, systems and kits for the detection of microorganisms of the  Yersinia  species including  Yersinia pestis . The disclosure relates to recombinant phage operable to infect a  Yersinia  microorganism, the phage comprising a detectable reporter. Detection systems of the disclosure may comprise a phage operable to infect a  Yersinia  microorganism, and may comprise a reporter nucleic acid expressible upon infection of a  Yersinia  microorganism by the phage. The system may be operable to detect the expression of the reporter. A detectable reporter may comprise any gene having bioluminescent, colorimetric and/or visual detectability. For example, a detectable reporter may comprise one or more luxAB genes detectable by emission, enhancement and/or change in spectrum of bioluminescent light. Live and infectious  Yersinia  microbes may be detected by the compositions, methods, systems and kits described herein.

PRIORITY

This application is a 371 U.S. national application of InternationalApplication Number PCT/US2009/043776 filed May 13, 2009, whichdesignates the United States, and claims priority to U.S. ProvisionalApplication Ser. No. 61/127,506, filed May 14, 2008. The contents ofwhich are hereby incorporated by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with government support under governmentcontract number 1R43AI082698-01, Plague related NIH SBIR Grant. The U.S.Government has certain rights in this invention.

FIELD OF THE DISCLOSURE

The present disclosure relates to compositions, methods, systems andkits for detection of microbes. In some embodiments, the compositions,methods, systems and kits of the disclosure are directed to thedetection and/or identification of biological pathogens of Yersiniaspecies (e.g., Yersinia pestis).

BACKGROUND OF THE DISCLOSURE

Yersinia pestis is classified by the Centers for Disease Control andPrevention (CDC) and the National Institutes of Health (NIH) as aCategory A priority bacterial pathogen that will most likely be used ina bioterrorist attack. Y. pestis is the etiological agent of the plague(Black Death), a transmissible disease that has been responsible formillions of deaths throughout the course of history. Typically, humanscontract plague after being bitten by a rodent flea that carries theplague bacterium or by handling an infected animal. Millions of peoplein Europe died from plague during the Middle Ages, when human homes andplaces of work were inhabited by flea-infested rats.

Although the natural occurrence of the disease is now relatively rare,the deliberate release of Y. pestis is a real threat. Dispersal willmost likely be in the form of an aerosolized release over a populatedarea. The first signs of an attack will be outbreaks of pneumonic plague1-4 days later. If left untreated, pneumonic plague is nearly alwaysfatal. Y. pestis may be transmitted from person to person. Transmissionmay occur through infectious respiratory droplets from pneumonic casesof the plague, or even from inhalation from contaminated clothes.

The use of Y. pestis as a biological weapon is not without precedent.The Tartars, during the siege of the Genoese-controlled Black Sea portof Kaffa, hurled plague-infected corpses over the city walls into thehuddled city. During World War II, the Japanese reportedly releasedplague-infected fleas over populated areas of China, which resulted insporadic plague outbreaks. Recent technological advances have enabled Y.pestis to be directly aerosolized, which is considered to be the mostlikely way the agent would be dispersed. The World Health Organization(WHO) estimates that an aerosolized release of 50 kg over a populatedcity could cause 150,000 cases of pneumonic plague and 36,000fatalities. Compounding this threat is the possibility of deliberatelyreleasing engineered antibiotic resistant strains which were reportedlyproduced in the former Soviet Union. Consequently, the potential forpublic disruption and panic would be severe.

SUMMARY

Accordingly, a need has arisen for compositions, methods, systems,and/or kits for easily and/or rapidly detecting microbes (e.g., Yersiniapestis) in a laboratory and/or in a non-laboratory setting.

The present disclosure relates, according to some embodiments, tocompositions, methods, systems, and/or kits for easy and/or rapiddetection of microbes (e.g., of the Yersinia sp.) in a laboratory and/orin a non-laboratory setting.

In some embodiments, the present disclosure relates to a phage operableto infect a Yersinia microorganism comprising a reporter. In someembodiments, the reporter may comprise a nucleic acid. The nucleic acidmay encode one or more detectable gene products. For example, thereporter may comprise a nucleic acid that, upon expression, leads to theproduction of one or more detectable products. Detectable gene productsmay include, for example, enzymes that catalyze bioluminescent reactions(e.g., encoded by luxAB) and/or fluorescent proteins (e.g., GFP, DsRed)that may be detected with a light detector. Detectable gene products mayinclude, for example, enzymes that catalyze reactions with coloredreactants and/or products (e.g., encoded by lacZ and/or gusA) that maybe detected colorimetrically.

The disclosure relates to phage that may infect a Yersiniamicroorganism. In some embodiments, the disclosure relates to a phage ofserovar 1, and/or serovar 2, and/or serovar 3, and/or serovar 4, or ofany serovar that Yersinia-specific phages may be categorized into. Insome embodiments, the phage may be a lytic phage, such as a φA1122phage. In some embodiments, the phage may be a temperate phage and maycomprise a L-413C phage.

The present disclosure also relates to a detection system comprising:(a) a phage operable to infect a Yersinia microorganism, comprising areporter configured and arranged to be expressed upon infection of theYersinia microorganism by the phage; and (b) a detector operable todetect reporter expression.

Expression of a gene, in some embodiments, may include transcriptionand/or translation. According to some embodiments, expression mayinclude post transcriptional and/or posttranslational modification(s) ofa gene product. In some embodiments, one may detect a detectable geneproduct that may be formed following phage binding and/or infection of aYersinia microorganism. For example, a detectable gene product mayinclude a product of transcription (e.g., an RNA), a product oftranslation (e.g. a peptide or a protein), and/or a product of posttranscriptional and/or posttranslational modification. A detectable geneproduct, in some embodiments, may include a product that may form as aresult of phage binding or infection which does not requiretranscription and/or translation.

In some embodiments, a reporter may comprise a nucleic acid. A reporternucleic acid, in some embodiments, may be operably linked to one or moreYersinia expression control elements that control the expression of oneor more detectable genes. Expression of a detectable gene may refer totranscription, and/or synthesis of RNA and/or stable accumulation of RNA(e.g., mRNA). Expression may also refer to translation of mRNA into apolypeptide as well as modification of such a polypeptide or protein byposttranslational mechanisms. For example, a Yersinia expression controlelement may comprise one or more transcriptional control elements(non-limiting examples include promoters (e.g., −10 box, −35 box,heat-shock promoters, etc.), enhancers, inducers, transcriptionalrepressors, transcriptional terminators, RNA processing or stabilizingelements, one or more translational control elements (non-limitingexamples include translation leader sequences, RNA processing site,effector binding site and stem-loop structure), one or moreposttranslational modifying elements, and/or combinations thereof.

In some embodiments of the disclosure, a reporter nucleic acid maycomprise one or more luxAB gene(s). LuxAB genes encode for an enzyme, aluciferase, that catalyzes the production of bioluminescent light thatmay be detected using a photodetector. Nucleic acids encoding luxABgenes may be derived from any organism of any species. For example, aluxAB from Vibrio harveyi, Xenorhadbus luminescens, V. fischeri,Photinus pyralis (firefly), Photobacterium sp., Photorhabdusluminescens, or any species expressing luxAB may be used. In someembodiments, one or more luxAB genes encoding mutations that emit lightat various (different) wavelengths may be used.

A nucleic acid construct, also referred to as a vector or a cassette,comprising a luxAB gene (or any other detectable gene) may haveexpression control elements, such as but not limited to the following:transcriptional control elements (for example, promoter elements such asbut not limited to those described above; enhancers; inducers;transcriptional repressors; and/or transcriptional terminators, etc.);translational control elements; posttranslational modifying elements;and/or combinations thereof that may control its expression. In someembodiments, one or more of the expression control elements may bederived from Yersinia sp. In some embodiments, one or more of theexpression control elements may require at least one regulatory moietyderived from a Yersinia organism for turning on the expression of theluxAB gene (or any other detectable gene). An exemplary regulatorymoiety derived from a Yersinia organism may be a protein (e.g., atrans-activating protein), a gene regulatory element (such as aribosome), an RNA, a DNA, a co-factor. In some embodiments, a nucleicacid construct having a luxAB gene (or any other detectable gene) may bedetectable once transformed or transfected into a Yersinia cell or in alive Yersinia cell.

In some embodiments, one or more promoters used to control theexpression of a detectable gene (such as luxAB, or a fluorescent proteingene) may be derived in their entirety from a native gene (such as froma Yersinia Sp.), or be composed of different elements derived fromdifferent promoters (from Yersinia sp. or those found in nature), or mayeven comprise synthetic DNA segments. The location of a detectable gene(i.e., such as but not limited to a luxAB gene, a fluorescent proteingene) in a nucleic acid construct, in accordance with the teachings ofthe present disclosure, may vary based on expression characteristicswhich may depend on the particular nucleic acid construct and/orexpression control sequences employed. One of skill in the art willrealize that such variations are all within the scope of the presentdisclosure.

In some embodiments, a reporter may comprise a nucleic acid encoding theluxCDE genes (from any species) in addition to a luxAB gene. LuxCDEgenes encode a fatty acid reductase complex that synthesizes fattyaldehydes which are substrates for the luminescence reaction. This maypartially and/or completely eliminate the need to add aldehydesubstrates to produce and/or detect bioluminescence.

A detection system of the disclosure may be configured to detect anyYersinia microorganism. In some embodiments, Yersinia pestis may bedetected. In some embodiments, other human pathogenic Yersiniamicroorganisms, for example, Yersinia enterocolitica, Yersiniapseudotuberculosis, and combinations thereof may be detected. Thedetection system of the disclosure may also detect Yersiniamicroorganisms that are pathogenic to other animals, birds, fish, andthe like. For example, Yersinia ruckeri (a fish pathogen) may bedetected.

In some embodiments, a detection system may include phage that mayinfect a Yersinia microorganism. According to some embodiments, anyphage that infects any Yersinia microorganism may be used. A phage mayinclude a phage of serovar 1, and/or serovar 2, and/or serovar 3, and/orserovar 4, or of any serovar or any other classification thatYersinia-specific phages may be categorized into, according to someembodiments. A detection system, in some embodiments, may include alytic phage (e.g., a φA1122 phage). In some embodiments, a detectionsystem may include a temperate phage (e.g., a L-413C phage).

The disclosure also relates to methods of detecting the presence of aYersinia microorganism in a test sample. In some embodiments, adetection method may comprise: a) providing a phage operable to infect aYersinia microorganism, wherein the phage comprises a reporterconfigured and arranged to be expressed upon infection of the Yersiniamicroorganism by the phage; b) contacting the test sample with the phageunder conditions that permits the phage to infect the Yersiniamicroorganism and express the reporter; and c) detecting expression ofthe reporter, if any, wherein detecting the reporter indicates that theYersinia microorganism is present in the test sample.

Methods of the disclosure may be configured to detect any Yersiniamicroorganism from a test sample that may be suspected of comprisingYersinia, according to some embodiments. For example, human pathogenicYersinia microorganisms such as Yersinia pestis, Yersiniaenterocolitica, Yersinia pseudotuberculosis, and combinations thereofmay be detected. Methods of the disclosure may also detect Yersiniamicroorganisms that are pathogenic to other animals, birds, fish, etc.,for example, Yersinia ruckeri (a fish pathogen) may be detected. Testsamples may be biological test samples collected from a human or ananimal or they may be non-biological samples. Biological samples mayinclude any sample derived from the body of an animal or human that maybe infected or is suspected of being infected. Non-biological samplesmay include a food sample, a water sample, an air sample, and the likemay be tested for the presence of a Yersinia microorganism.

In some embodiments, detecting expression of a reporter may comprisedetecting bioluminescence. Detecting bioluminescence may compriseproviding a substrate intended to react with a luxAB gene product. Oneexemplary substrate may comprise an aldehyde such as n-decanal. Inembodiments where a luxCDE gene may also be present in a reporter system(on the same or separate reporter), detecting bioluminescence may notrequire the provision of an aldehyde substrate and/or may requireproviding a smaller amount of an aldehyde substrate as compared to whena luxCDE gene may not be present in the reporter system.

The present disclosure also relates to kits for detecting the presenceof a Yersinia microorganism. A kit, in some embodiments, may be used ina laboratory and/or outside of a laboratory setting. In someembodiments, a kit according to the disclosure may comprise a) a phageoperable to infect a Yersinia microorganism, comprising a reporterconfigured and arranged to be expressed upon infection of the Yersiniamicroorganism by the phage, in a suitable container; and b) one or morecontainers to mix the phage with a test sample that may comprise theYersinia microorganism. A kit, according to some embodiments, maycomprise a detector substrate in a suitable container. In someembodiments, a kit may comprise a bioluminescence detector (e.g., aphoton detector; a detector configured and arranged to detect differentwavelengths of bioluminescent light).

In some embodiments, a kit may comprise one or more Yersinia specificphages. Any phage that infects any Yersinia microorganism may be used.In some embodiments a kit of the disclosure may comprise one or moreYersinia microorganism as a control.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the disclosure may be understood by referring, inpart, to the present disclosure and the accompanying drawings, wherein:

FIG. 1 illustrates a schematic of a luxAB expression cassette and Pro1conserved nucleotides (arrow denotes direction of transcription),according to a specific example embodiment of the disclosure;

FIG. 2 illustrates a schematic of a Yersinia shuttle vector andhomologous recombination process based on a double crossover event,according to a specific example embodiment of the disclosure;

FIG. 3 shows integration of luxAB into φA1122 at the correct site in thephage genome: PCR analysis was performed in the absence of template(lane 1), with the wild-type φA1122 phage (lane 2) or with therecombinant φA1122::luxAB phage (lane 3), PCR products for the 5′junction, 3′ junction, and luxA of 591, 521, and 163 bp, respectivelyare seen in lane 3 and not in the control lanes indicating the presenceof luxA and integration of the luxAB into the φA1122 genome at theexpected location, according to a specific example embodiment of thedisclosure; and

FIG. 4 illustrates detection of Y. pestis by φA1122::luxAB measured asbioluminescence (RLU) over time following the addition of 2% n-decanal,according to a specific example embodiment of the disclosure.

DETAILED DESCRIPTION

Current biological detection methods for the detection of Yersiniapestis may be time consuming and/or expensive and/or may requireexpensive laboratory equipment and/or expertise. Methodologies for rapidand sensitive Y. pestis detection are critically needed to combat thethreat of deliberate release of this pathogen. Phage specific lysisassays, using Y. pestis specific phage, may be used as a diagnosticstandard for the confirmed identification of Y. pestis. However,laboratory-based methods may require elaborate sample processing, and/orextensive incubation periods, and/or 18-24 hours to complete.Immunological methods using fluorescent-antibodies specific to Y. pestisF1 envelope glycoprotein and/or capsular antigen may be used. However,immunological methods may require long incubation and reaction periods,expensive reagents, and/or a laboratory setting to perform.

Rapid and sensitive detection methodologies may contribute to lowermorbidity and save lives (e.g., where a bioterrorist attack affects alarge population at once). Some embodiments of the present disclosurerelate to compositions, methods, systems and kits for detection ofmicrobes (e.g., Y. pestis), that may provide desirable speed andsensitivity and may be used in and/or outside of a laboratory.

The present disclosure relates, in some embodiments, to biologicaldetection compositions, methods, systems and/or kits for rapid detectionof a bacterial cell such as a Yersinia sp. cell. There are about 11named species in the genus Yersinia of which three species are known tobe human pathogens: Yersinia pestis, Yersinia enterocolitica, andYersinia pseudotuberculosis. The compositions, methods, systems and/orkits of the present disclosure, in some embodiments, may be used, forthe detection of one or more Yersinia species exemplified innon-limiting examples by the human pathogenic strains Yersinia pestis,Yersinia enterocolitica, Yersinia pseudotuberculosis, as well asmutations and genetically engineered variants thereof, etc. Withoutlimiting any embodiment of the disclosure to a particular Yersiniaorganism, mechanism of action, symptom(s), and/or modes of diseasetransmission some Yersinia-mediated diseases and conditions aredescribed.

Y. pestis is the etiologic agent of plague which is a zoonotic diseaseaffecting rats and other rodents. Y. pestis may be transmitted fromanimal to animal by fleabites, which may also be the most common routeof transmission to humans. Y. pestis-infected flea bites, leads to themigration of the bacterium to the lymph nodes and bubonic plaguedevelops 2-8 days which is characterized by fever, chills, weakness andthe development of swollen lymph nodes or buboes. In a minority ofcases, the fleabites develop into septicemia without a bubo, oroccasionally into pneumonic plague. The occurrence of the plague is rarein the U.S. with only an average of 5-15 cases reported each year,mostly in rural areas. The epidemiology of the disease, however, may bevery different during deliberate release, because an aerosolized attackof Y. pestis would lead to a massive outbreak of pneumonic plague. Earlydetection and diagnosis would be desirable since the only indications ofan attack would be outbreaks of illness 1-4 days later presenting assevere pneumonia. Pneumonic plague is nearly always fatal if untreated.

Y. pseudotuberculosis and Y. enterocolitica are enteropathogenicYersinia strains that may be transmitted orally and cause a range ofgastrointestinal diseases collectively referred to as yersiniosis. Y.enterocolitica generally infects young children and some associatedsymptoms include fever, abdominal pain, and diarrhea, which is oftenbloody. Symptoms typically develop 4 to 7 days after exposure and maylast 1 to 3 weeks or longer. Symptoms in older children and adultsinclude right-sided abdominal pain and fever which may be confused withappendicitis. In a small proportion of cases, complications such as skinrash, joint pain, or spread of bacteria to the bloodstream may occur. Y.pseudotuberculosis is the closest genetic relative to Y. pestis but maybe distinguished from the plague bacteria by its clinical manifestationsand by laboratory test results. Y. pseudotuberculosis relatedgastrointestinal diseases are relatively rare but human infectionstransmitted via contaminated water and foods have been reported.

Embodiments of the present disclosure provide for compositions, methods,systems and/or kits for detection of bacteria of any Yersinia sp. andmay be useful in detecting the pathogenic strains of Yersinia thatafflict humans and/or animals.

In some embodiments, the compositions, methods, systems and/or kits ofthe disclosure, comprise a bacteriophage that is operable to infect aYersinia microorganism. In some embodiments, one or more bacteriophagespecific to Yersinia (e.g., Y. pestis) may be used. Y. pestis specificphage may be placed into four serovars based on their immunogenicity:(i) serovar 1 consists of lytic phages and is exemplified by the plaguediagnostic phage φA1122; (ii) serovar 2 is exemplified by the temperatephage L-413C; (iii) serovar 3 is exemplified by a temperate phage,termed P, and serovar 4 is exemplified by the phages Tal and 513. Thelytic phages of serovar 1, and in particular, the ‘plague diagnostic’phage φA1122, have a broad host strain infectivity and speciesspecificity. These phages all have isometric hexagonal heads and short(13-42 nm) non-contractile tails. They belong to the family Podoviridaeand are closely related to the Escherichia coli phages T3 and T7. TheφA1122 genome has recently been sequenced and found to consist of 37,555bp, encoding 51 predicted gene products, and a nucleotide identity of89% to the E. coli phage T7 (GenBank Accession No. AY247822 and GenBankAccession Number NC_(—)004777 as of 11-APR-2006). Phage φA1122 is‘specific’ to Y. pestis species with the exception of the closelyrelated species Yersinia pseudotuberculosis. However, temperature may beused to differentiate the two species since the phage does not grow onY. pseudotuberculosis at 20° C. Moreover, the phage has a very broadhost range within the species Y. pestis. According to the CDC, theφA1122 phage can grow and lyse on all but two of thousands of naturalisolates of Y. pestis within the CDC collection. Consequently, due toits specific and broad strain infectivity, φA1122 is used by the CDC,the WHO, and the U.S. Army Medical Research Institute of InfectiousDiseases as a diagnostic standard (lysis assay) for the confirmedidentification of Y. pestis. In some embodiments of the compositions,methods, systems and/or kits of the disclosure, the bacteriophageoperable to infect a Yersinia microorganism may comprise a reporter. Insome embodiments, the reporter is a detectable reporter. In someembodiments of the disclosure, a lytic phage, e.g., a φA1122 phage, maybe used to detect Yersinia microorganisms. In some embodiments of thedisclosure, a temperate phage, e.g., a L-413C phage, may be used todetect Yersinia microorganisms.

In some embodiments, a φA1122 phage may comprise a reporter. In someembodiments, a reporter may comprise a nucleic acid which leads to theproduction of a detectable gene product. In some embodiments, adetectable gene product may comprise a protein that is encoded by theluxAB genes from Vibrio harveyi (GenBank Accession No. E12410, version1, last updated Apr. 20, 2006). In some embodiments, a phage may be agenetically engineered phage comprising nucleic acids encoding adetectable gene product, e.g., a luxAB gene that encodes a luciferaseenzyme.

In some embodiments, a detectable gene product may comprise or consistof a luciferase enzyme. Contacting a luciferase enzyme with a suitableluciferin substrate may produce bioluminescence. Substrates for aluciferase enzyme may comprise an aldehyde (e.g., n-decanal). In someembodiments, detection of bioluminescence upon binding or infection ofthe bacterial cell by the phage detects the presence of the Yersiniamicroorganism. The bioluminescent light signal may be visualized by asimple hand-held photon-detection device and no processing of the samplemay be required. In some embodiments, the light signal detected may beanalyzed for different wavelengths. In some embodiments the detection ofthe detectable reporter gene product may be by PCR or immunologicalmethods.

In some embodiments, the detection of bioluminescence may be achieved ina time of less than 30 minutes following infection with a recombinantphage of the disclosure. In some embodiments, the detection ofbioluminescence, and hence of an Yersinia microbe, may be in a time lessthan 25 minutes, less than 20 minutes, less than 18 minutes, less than17 minutes, less than 16 minutes, less than 15 minutes, less than 14minutes, less than 13 minutes, less than 12 minutes, less than 11minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes,less than 7 minutes, less than 6 minutes, less than 5 minutes, less than4 minutes less than 3 minutes, less than 2 minutes to less than 1minute, following infection with a recombinant phage of the disclosure.

The lux genes control bioluminescence in a wide variety of speciesincluding marine and terrestrial species such as bacteria,dinoflagellates, fungi, fish, insects, shrimp, and squid. Cloning andexpressing lux genes from different species have led to significantadvances in understanding the molecular biology of bioluminescence. Luxoperons may have a common gene organization of luxCDAB(F)E, with luxABcoding for the enzyme luciferase and luxCDE coding for the fatty acidreductase complex responsible for synthesizing fatty aldehydes which aresubstrates for the luminescence reaction. However, significantdifferences exist in their sequences and properties as well as in thepresence of other lux genes (such as lux I, R, F, G, and H). In someembodiments, a luciferase-encoding nucleic acid (e.g., luxAB from anyspecies) may also be used in the compositions, methods, systems and/orkits of the disclosure. For example, a luxAB from Xenorhadbusluminescens may be used. Other non-limiting examples include a luxABfrom V. fischeri, Photinus pyralis (firefly), Photobacterium sp., andPhotorhabdus luminescens.

In some embodiments, the phage operable to infect a Yersiniamicroorganism, may comprise in addition to a reporter, nucleic acidsencoding for the luxCDE genes (from any species), which encode a fattyacid reductase complex that may synthesize fatty aldehydes which aresubstrates for the luminescence reaction. This may eliminate the need toadd aldehyde substrates to detect bioluminescence.

Expression of recombinant φA1122 luxAB and light production may requirea phage-infected bacterial cell being detected to have an activemetabolism. In some embodiments, a phage reporter gene may be under thecontrol of one or more transcriptional elements (such as but not limitedto, promoters, enhancers, repressors, transcriptional terminators, etc.)and/or translational elements (such as but not limited to, ribosomebinding sites). Posttranslational modifying elements may also bedesirable. A reporter gene may comprise a nucleic acid encoding adetectable gene product as well as transcriptional and/or translationalcontrol elements for expression of the detectable gene product.Bacterial cellular machinery may be desirable for expression of areporter gene comprising one or more bacterial transcriptional and/ortranslational elements. In some embodiments, production of thedetectable gene product by the reporter nucleic acid may be dependent onone or more components of the bacterial cell that the phage isinfecting. For example, expression of the reporter may indicate thepresence of live and infectious bacteria as well as bacteria of aspecific species. Since only viable cells produce a light signal, theseexample compositions, methods, systems and/or kits of the disclosure ofthe present disclosure detect viable and infectious bacterial cells.This is a distinct advantage over PCR detection methodologies and theimmunological F1 antigen tests which detect the presence of Y. pestis,but yield no information as to whether the Y. pestis cells are viableand infectious.

In some embodiments, the present disclosure, relates to methods forpreparing a bacteriophage configured and arranged to detect a microbe.For example, a Y. pestis luxAB reporter phage may be generated using thediagnostic plague phage φA1122. LuxAB may be cloned into an expressioncassette under the transcriptional and translational control ofpreferred Yersinia expression sequences. The expression cassette may beflanked by φA1122 phage DNA to allow homologous recombination of theexpression cassette into the phage DNA. LuxAB may be integrated into anon-coding region of the φA1122 genome by homologous recombination basedon a double cross over event. Recombinant φA1122::luxAB may beidentified and isolated based on the ability of infected cultures toemit light. LuxAB integration may be verified by diagnostic agarose gelelectrophoresis and PCR. The ‘fitness’ of the recombinant phage may becompared to the wild-type phage.

The sensitivity of the bioluminescence assay may be from about 1 CFU/mLto about 50,000 CFU/mL or about 100 CFU/mL to 1000 CFU/mL or about 1CFU/mL to about 100 CFU/mL. In some embodiments, the sensitivity may beabout 1 CFU/mL, 2 CFU/mL, 3 CFU/mL, 4 CFU/mL, 5 CFU/mL, 6 CFU/mL, 7CFU/mL, 8 CFU/mL, 9 CFU/mL to about 10 CFU/mL.

In some embodiments, the sensitivity may be about 1 CFU/mL, about 10CFU/mL, about 20 CFU/mL, about 30 CFU/mL, about 40 CFU/mL, about 50CFU/mL, 60 CFU/mL, about 70 CFU/mL, about 80 CFU/mL, about 90 CFU/mL toabout 100 CFU/mL. In some embodiments, the sensitivity may be from about100 CFU/mL to about 1000 CFU/mL and may be about 100 CFU/mL, about 200CFU/mL, about 300 CFU/mL, about 400 CFU/mL, about 500 CFU/mL, about 600CFU/mL, about 700 CFU/mL, about 800 CFU/mL, about 900 CFU/mL, to about1000 CFU/mL. In some embodiments, the sensitivity may include values inbetween the ranges listed above.

In some embodiments, the sensitivity of the assay may be about 1000CFU/mL to about 50,000 CFU/mL, and may be about 1000 CFU/mL, about 2000CFU/mL, about 3000 CFU/mL, about 4000 CFU/mL, about 5000 CFU/mL, about6000 CFU/mL, about 7000 CFU/mL, about 8000 CFU/mL, about 9000 CFU/mL,about 10,000 CFU/mL, about 15,000 CFU/mL, about 20,000 CFU/mL, about25,000 CFU/mL, about 30,000 CFU/mL, about 35,000 CFU/mL, about 40,000CFU/mL, about 45,000 CFU/mL to about 50,000 CFU/mL.

In some embodiments of the compositions, methods, systems and/or kits ofthe disclosure, a reporter may comprise a nucleic acid which leads tothe production of a detectable gene product. In some embodiments, thereporter may comprise nucleic acids that lead to the production of afluorescent protein such as a green fluorescent protein (GFP), which maybe detected as a green fluorescent light when exposed to UV light. Insome embodiments, the reporter may comprise nucleic acids that lead tothe production of a GFP, a red fluorescent protein (DsRed), or a yellowfluorescent protein or mutations and variants thereof.

In some embodiments, the reporter may comprise nucleic acids that leadto the production of an ice nucleation gene (inaZ). In some embodiments,the reporter may comprise nucleic acids that lead to the production ofthe beta-glucuronidase (gusA), which may be detected by colorimetricenzyme assay of cell extracts or indicator plates.

In some embodiments, the reporter may comprise nucleic acids that encodea lacZ gene, which encodes an enzyme B-galactosidase. Cells expressingB-galactosidase turn blue color when grown on a medium that contains theB-galactosidase substrate (e.g., the analog X-gal) which may be detectedcolorimetrically.

In some embodiments, the reporter may comprise nucleic acids that encodeselectable-marker reporter which may confer an antibiotic resistantphenotype on the bacteria expressing the marker gene, e.g., a reportermay encode a chloramphenicol acetyltransferase (CAT) gene which confersresistance to the antibiotic chloramphenicol.

In some embodiments, the present disclosure relates to compositions,methods, systems and/or kits for detecting the presence of Yersiniabacterial cells that may not (e.g. do not) require sample processing,extensive incubation periods, or a laboratory environment. Recombinantphage cells may be mixed with a test sample suspected of comprising aYersinia microorganism and subsequently analyzed for bioluminescence. Asuitable aldehyde substrate (e.g. n-decanal) may be also mixed in toobtain and/or enhance bioluminescence.

The test sample suspected of comprising a Yersinia microorganism may beany kind of a sample including biological samples such as blood, serum,fluid from bubos, nasal fluids, respiratory tract washes, nasal swabs,throat swabs, mucous, urine, stools, or any other bodily fluids. Thetest sample also may be a non-biological sample such as a an air sample(e.g., air sample suspected of having aerosolized Yersinia pestis); anenvironmental sample such as a soil or a water sample; a food sample,including processed and cooked foods, raw vegetables, fruit, water,diary products, etc. Air samples may be collected by trapping a samplevolume of air (from a specific location) in a tube, packet or containeror by any other method known in the art to collect air samples.

Compositions, methods, systems and/or kits, according to someembodiments of the disclosure, may be configured to permit rapiddetection of a microorganism such as a Yersinia microorganism. Forexample, a Yersinia microorganism may be detected in less than abouttwelve (12) hours, less than about ten (10) hours, less than about eight(8) hours, less than about six (6) hours, or less than about four (4)hours. A target microorganism may be detected in less than about three(3) hours, less than about two (2) hours, or less than about one (1)hour. A target microorganism may be detected in less than about fortyminutes, less than about thirty minutes, less than about twenty minutes,less than about fifteen minutes, less than about thirteen minutes, lessthan about twelve minutes, less than about eleven minutes, less thanabout ten minutes, less than about nine minutes, less than about eightminutes, less than about seven minutes, less than about six minutes,less than about five minutes, less than about four minutes, less thanabout three minutes or less than about two minutes. The time requiredfor detection may be a function of the time required for infection,and/or reporter expression and detection.

The present disclosure, in some embodiments, also relates to kits fordetecting Yersinia microorganisms. A kit, in some embodiments, mayprovide components necessary and/or desired for detecting a Yersiniamicroorganism in a test sample (e.g., a biological sample obtained froma patient or animal). Non-biological samples may also be tested for thepresence of Yersinia microorganisms to detect contamination and theseinclude air samples, food samples including processed and cooked foods,raw vegetables, fruit, diary products, drinks, water and the like. Insome embodiments Yersinia species that cause gastrointestinal disorders,(e.g., Y. pseudotuberculosis and/or Y. enterocolitica), may be detectedfor preventing food/water borne illnesses. In some embodiments, a kitmay comprise compositions and/or materials for detecting Y. pestis inbiological samples and/or from non-biological samples.

A diagnostic kit, according to some embodiments, may comprise a) agenetically engineered phage operable to infect a Yersiniamicroorganism, wherein the phage comprises a reporter gene that isdetectable only after phage infection of a Yersinia microorganism; b) adetector substrate that forms a detectable substrate upon expression ofthe reporter gene; and c) one or more containers to contact (e.g., mix),the phage with a test sample that may comprise a Yersinia microorganismand detector substrate. Each component of the kit may be contained in asuitable container means such as a vial, tube etc. and may be comprisedin suitable solvents, buffers, or reagents. Alternatively somecomponents may be present in a dry, powdered or lyophilized form. Insome embodiments, a kit may also include suitable solvents, buffersand/or reagents required to reconstitute one or more component(s) asrequired.

In some embodiments, a kit of the present disclosure may comprise a) agenetically engineered φA1122 phage operable to infect Y. pestis andcomprising a luxAB reporter gene φA1122::luxAB); b) a detector substratefor example, (e.g., an aldehyde such as n-decanal), that may react withthe luxAB gene product to produce a detectable product (e.g.bioluminescent light); c) optionally a means for detecting thebioluminescent light. Each component may be packaged in suitablebuffers, solutions or reagents and/or may be available as dry orlyophilized form.

In some embodiments, a kit according to the present disclosure maycomprise a) a genetically engineered phage (e.g., φA1122) operable toinfect Y. pestis, comprising a luxAB reporter gene (such asφA1122::luxAB) and a luxCDE gene; b) a means for detectingbioluminescent light. Such a kit may optionally need small amounts of adetector substrate such as an aldehyde such as n-decanal, in case theluxCDE genes do not produce sufficient substrate that may react with theluxAB gene product to produce detectable bioluminescent light. Eachcomponent may be packaged in suitable buffers, solutions or reagentsand/or may be available as dry or lyophilized form.

A kit, in some embodiments, may comprise one or more standard samplescomprising Yersinia sp., for example, Y. pestis, for providing ameasuring standard. Phage, (e.g., recombinant phage) may be resistant toenvironmental extremes and/or may be stored for months or years withouta significant loss in phage infectivity. Bacterial cells however, mayloose their viability and/or susceptibility to phage infection afterstorage for long periods of time. Thus, storage periods and storageconditions for components of a kit may vary.

A container may include any vessel into which a material may be placed(e.g., a vial, test tube, flask, bottles, syringe, pipette, and/or plateother container means. The individual containers of a kit may bemaintained in close confinement (e.g., for commercial sale). Suitablelarger containers may include injection or blow-molded plasticcontainers into which the desired vials are retained. Instructionsand/or safety information may be provided with a kit.

Additionally, a bioluminescence detector such as a simple photodetectormay be provided. A skilled artisan, having the benefit of the presentdisclosure, will recognize that any photodetector known in the art maybe suitably used with the compositions, methods, detection systemsand/or kits of the present disclosure.

As will be understood by those skilled in the art who have the benefitof the instant disclosure, other equivalent or alternative compositions,devices, methods, systems and/or kits for detecting Yersiniamicroorganisms or other bacterial microorganisms using bacteriophagescan be envisioned without departing from the description containedherein. Accordingly, the manner of carrying out the disclosure as shownand described is to be construed as illustrative only. Persons skilledin the art may make various changes in the shape, size, number, and/orarrangement of parts without departing from the scope of the instantdisclosure. For example, the location of a detectable reporter gene inthe phage may be changed, and/or one or more different promoters and/orother expression/regulatory control sequences from those expresslydescribed herein may be used. In some embodiments, a Yersiniaexpression/regulatory control sequence and/or a variant of a Yersiniaexpression/regulatory control element (such as promoter), and/or aexpression/regulatory control element having a synthetic orsemi-synthetic component may be used in accordance to the teachingsherein. In another example, the type of a detectable reporter gene inthe phage may be changed.

In addition, the size of a detection method, system and/or kit may bescaled up or down to suit the needs and/or desires of a practitioner.Also, where ranges have been provided, the disclosed endpoints may betreated as exact and/or approximations as desired or demanded by theparticular embodiment. In addition, it may be desirable in someembodiments to mix and match range endpoints. A composition, methodsystem or kit may be configured and arranged to be disposable,serviceable, interchangeable, and/or replaceable. These equivalents andalternatives along with obvious changes and modifications are intendedto be included within the scope of the present disclosure. Accordingly,the foregoing disclosure is intended to be illustrative, but notlimiting, of the scope of the disclosure as illustrated by the followingclaims.

EXAMPLES

Some specific example embodiments of the disclosure may be illustratedby one or more of the examples provided herein. Although most of theembodiments here are described with reference to phage φA1122 and aluxAB reporter gene, it will be understood that these examples areprovided for illustrative purposes only. They are not to be construed aslimiting the scope or content of the disclosure in any way and any phagecompatible to infect a Yersinia species as well as any detectablereporter gene may be used, in light of the embodiments of thisdisclosure.

Example 1 Y. pestis Strain and Phage Propagation

Y. pestis specific diagnostic phage φA1122 may be obtained from the CDC.The attenuated Y. pestis A1122 strain may be obtained from BeiResources(Bei#NR15, NIH/ATCC Biodefense and Emerging Infections ResearchResources Depository). The Y. pestis A1122 strain is an excluded selectagent strain which lacks the 75 kb low-calcium response (Lcr) virulenceplasmid, and is thus irreversibly attenuated. A similar Lcr negativestrain (Tjiwidej S) has been routinely used as a live vaccine in humansin Java indicating that the strain poses little to no threat to publichealth. Nevertheless, experiments involving Y. pestis A1122 may beperformed under BSL2 conditions as recommended by BeiResources. Y.pestis A1122 may be grown on brain heart infusion (BHI) agar and liquidbroth at 30° C. Clonal stocks of φA1122 phage may be prepared fromsingle plaques. Y. pestis A1122 may be prepared by growing the cells inBHI media at 30° C. until an OD₆₀₀ of 0.6 is reached. The cells may beharvested by centrifugation at 4,000×g for 10 min and resuspended in BHIto an OD₆₀₀ of 2.0. Cells (100 μl) may be mixed with an equal volume ofthe phage preparation and incubated at room temperature for 10 min toallow pre-absorption of the phage to the bacteria. A low MOI(multiplicity of infection) may be used to select for cells that areinfected by a single phage using the agar overlay method. Thephage/bacteria mixture may be added to pre-warmed (47° C.) ‘molten’ BHIcontaining 0.7% agar, mixed gently, and poured over pre-warmed BHI agarplates. The plate may be left on the bench until the agar solidifies,and then incubated upside down at 30° C. overnight. Presence of plaquesare indicative that phage are present.

To generate a phage stock, a distinct clonal plaque may be picked with asterile Pasteur pipette and propagated on Y. pestis A1122. The phage maybe amplified on progressively increasing culture volumes ofexponentially growing cells in BHI media at 30° C. After each overnightgrowth, the cultures may be centrifuged and the supernatants passedthrough a 0.22 μm filter. The phage stock may be concentrated accordingto Carlson 2005. Sodium chloride (NaCl) may be added (at 4° C. withmixing) to the phage preparation to give a final concentration of 0.75 Mand stored on ice for 60 min. The NaCl dissociates phage from thebacterial debris and improves polyethylene glycol (PEG) mediatedprecipitation in the subsequent steps. PEG 6000 may be added to a finalconcentration of 10%. After 4 h at 4° C., the phage may be collected bycentrifugation at 11,000×g for 20 min at 4° C. and the resulting phagepellet may be carefully reconstituted with SMC buffer (50 mM Tris-HCl[pH7.5], 0.1 M NaCl, 8 mM MgSO₄.7H₂O, 0.01% gelatin supplemented with 5mM CaCl₂) overnight at 4° C. The resulting phage preparation may betittered using the agar overlay technique and stored at 4° C. untilneeded.

Example 2 Construction and Design of a luxAB Expression Cassette

LuxA and luxB may be PCR-amplified using the proofreading thermostableenzyme PfuUltra (Stratagene) and pQF110 (ATCC77113, contains luxAB) astemplate. The PCR primers may be designed to contain restrictionendoculease sites for directional cloning into the corresponding sitesof pBluescriptSK⁻ (Stratagene). The 5′ primers may contain a consensusribosome binding site (TAAGGAGGTAAAAAA(ATG)) (SEQ. ID. NO: 1) which hasbeen shown to mediate efficient translation initiation in Gram-negativeEnterobacteriaceae species. The luxA and luxB may be sequentially clonedinto pBluescriptSK⁻ (to create pluxABSK⁻) by standard cloningmethodology, and transformed into the propagating strain E. coli ER2738.Diagnostic restriction endonuclease analysis and agarose gelelectrophoresis may be used to verify that the correct clone has beenselected. The sequence of the PCR-amplified luxA and luxB genes may beverified by deoxy dye terminator sequencing.

A designed gram-negative Yersinia promoter (Pro1), based on conservedgram-negative promoter elements, may be used to drive luxA and luxBexpression. The Pro1 promoter may contain the following conservedelements/nucleotides: (i) the −35 (TTGACA) (SEQ. ID. NO: 2) and −10(TATAAT) (SEQ. ID. NO: 3) hexanucleotide core elements, and (ii) 5 Aresidues upstream of the −35 region, i.e., AAAAA (SEQ. ID. NO: 4). Thedesigned promoter may be functional in both E. coli, and Y. pestis, andbe highly expressed. A highly expressed promoter may lead tooverexpression of the luxAB, high levels of bioluminescence, andpotentially a high sensitivity of detection. Pro1 may be cloned upstreamof luxA and luxB. To ensure efficient processing and to prevent runawaytranscription (into the neighboring phage genes), the transcriptionalterminator TL17 may be cloned downstream of the luxA and luxB genes(FIG. 1). The identity of the Pro1 and TL17 sequences may be verified bydeoxy dye terminator sequencing.

Example 3 Generation of the φA1122 Targeting Vector for HomologousRecombination

The φA1122 phage has recently been sequenced (GenBank Accession No.AY247822 and GenBank Accession No. NC_(—)004777), making geneticmanipulation of the phage more readily achievable. The phage genomeconsists of 37,555 bp, with 51 predicted gene products originating from46 distinct open reading frames. The φA1122 phage is very closelyrelated to the coliphage T7 (and to a lesser extent T3) sharing anucleotide identity of about 89%. A strategy involving direct cloning ofthe luxAB cassette into the φA1122 genome may not be pursued since thegenome is large (˜37 kb) making cloning difficult, and because it lacksappropriate restriction endonuclease sites. Therefore, the luxABcassette may be integrated into the φA1122 phage genome by homologousrecombination based on a double cross over event. The luxAB cassette maybe targeted for integration at two different sites in the phage genomeusing two slightly different approaches: (i) the luxAB cassette may beintegrated by insertion into the non-coding, intergenic region betweenthe ‘early’ genes gp1.3 and pg1.5. Adding the luxAB cassette (˜2 kb)into a non-coding region may not disrupt endogenous phage gene function,however, the phage genome may increase in size from ˜37 kb to ˜39 kb,and (ii) the φA1122 phage gene gp5.5 and surrounding non-coding DNA maybe replaced by the luxAB cassette. Gene gp5.5 may not play an essentialrole in phage propagation based on the observation that mutants of theT7 gene 5.5 homolog are still functionally viable, albeit with a reducedplaque size. Replacement of the gp5.5 with the luxAB cassette, ratherthan insertion, may limit the increase in size of the recombinant phagegenome, thereby reducing the risk of producing defective phage.Recombinant φA1122::luxAB generated by each approach may be compared forsimilarity in robustness and fitness to wild-type phage.

φA1122 DNA may be isolated from clarified lysates using a commerciallyavailable phage DNA isolation kit (Qiagen #12523). 500 bp fragmentsencompassing the 5′ and 3′ flanking φA1122 sequences may bePCR-amplified using φA1122 DNA as template and cloned into the pACYC184(New England Biolabs E4152S, GenBank Accession Number X06403). pACYC184is a multicopy E. coli/Yersinia shuttle vector containing multiplecloning sites, and the chloramphenicol or tetracycline resistance markerfor antibiotic selection. The luxAB expression cassette may be clonedinto an internal restriction site, and be flanked by the φA1122 DNA tocreate pluxABACYC184 (FIG. 2). FIG. 2 illustrates a schematic of aYersinia shuttle vector and homologous recombination process based on adouble crossover event. In FIG. 2, the approach of homologousrecombination by replacement of the non-essential gene gp5.5 usingflanking 5′ (gp5.0) and 3′ (gp5.7) homologous phage DNA is depictedaccording to some embodiments of the disclosure.

Prior to transforming the plasmids into Y. pestis, the plasmids may bepassaged through E. coli SCS110 (Stratagene) in order to generateplasmid DNA free of Dam and Dcm methylation and overcome possiblerestriction/modification when introducing foreign DNA into Yersinia. Theattenuated Y. pestis A1122 strain may be electroporated with pACYC184(control plasmid) and pluxABACYC184 and transformants may be selected onBHI agar supplemented with antibiotic. To analyze whether the luxABexpression vector is functional and produces light in Y. pestis, theresulting colonies may be examined under dark field illumination.Luminescent colonies may not be obtained for the control Y. pestisstrain harboring the empty control plasmid (pACYC184), however,luminescent colonies may be readily evident for Y. pestis harboringpluxABACYC184. This is an indicator that Y. pestis may be successfullytransformed with a functioning luxAB cassette.

Example 4 Homologous Recombination

Homologous recombination between phage and plasmid DNA based on a doublecrossover event may be used to integrate luxAB into the phage genome(FIG. 2). To allow for multiple rounds of phage propagation, φA1122 maybe introduced into Y. pestis A1122 harboring pluxABACYC184 by phageinfection using the agar overlay technique. Following infection andovernight growth, phage from multiple plates exhibiting confluent lysismay be eluted with 10 ml of SM buffer and filter sterilized. The titerof the phage lysates may be determined.

Example 5 Identification of Recombinant φa1122::luxAB Phages

φA1122::luxAB phage may be identified and isolated (also see Example 12and FIGS. 3-4). Mixed phage lysates, containing predominantly wild-typeφA1122 and a small number of φA1122::luxAB phages, may be used to infectY. pestis A1122 using the agar overlay technique with the followingmodifications: (i) 24×24 cm Petri dishes may be used instead of the‘standard’ 10×15 cm to allow more plaques to be screened per plate, and(ii) a high number of phage may be used for infection in combinationwith a short (10 h) overnight incubation to allow for the maximum numberof small and nearly confluent plaques per plate. Up to 25 plates may bescreened, each containing ˜3,000 to 5,000 plaques per plate. The platesmay be screened immediately following the addition of decanal vapor (2μl placed on the lid of the dish) under dark field illumination. For ahigh frequency of recombination at least 1 to 2 plates may contain asmall, but detectable light signal. Phage from the positive plates maybe eluted with 20 ml of SM buffer, filter sterilized and used forre-infection of Y. pestis A1122 using the agar overlay technique. Thisscreening process may be repeated until distinct individual plaquesemitting a bioluminescent phenotype may be picked and isolated. Hightiter φA1122::luxAB lysates may be prepared as described in Example 1.

Example 6 Analysis and Verification of the φA1122::luxAB RecombinantPhage

To confirm that integration was accomplished through a double crossoverevent, and to verify that the plasmid DNA backbone was not integratedinto φ1122::luxAB, phage DNA may be isolated and analyzed by diagnosticrestriction agarose gel electrophoresis and PCR (also see Example 12 andFIGS. 3-4). Diagnostic restriction digests, based on the known φA1122and luxAB sequences, may verify the presence of luxAB and the absence ofplasmid sequence. PCR analysis may also be used to verify the presenceof luxAB and absence of plasmid DNA using primers specific for luxAB (asdescribed in Example 2) and primers specific to pACYC184, respectively.To analyze whether the luxAB integration occurred at the correctpredicted site in the φA1122 genome, primers may be designed to spaneither the 5′ or 3′ integration junctions. Each primer set may bedesigned with one primer binding within the luxAB recombination cassetteand one primer binding external to the recombination cassette (in thephage DNA). PCR analysis using these ‘integration junction’ primers mayindicate that the luxAB cassette has integrated at the correct predictedsite. The phage lysates may also be treated with DNase-1 (digestsplasmid DNA but not ‘protected’ phage DNA) and subsequently used forphage infection; if DNase 1-treated cell free phage supernatants areable to transduce a bioluminescent phenotype to Y. pestis A1122, thismay collectively indicate that intact recombinant φA1122::luxAB phagehave been generated.

Foreign DNA may be inserted into phage without any loss of function,however, this may be dependent on phage type, the size of fragmentinserted, the orientation, and the site of insertion. Therefore, the‘fitness’ of the recombinant φA1122::luxAB phage may be compared to thewild-type φA1122 phage. Early exponential phase (OD₆₀₀ of 0.1) Y. pestiscells may be infected with the wild-type and recombinant phage at an MOIof 0.01 and incubated at 30° C. in BHI media. Following 6 h of growth,the cultures may be centrifuged, and the resulting supernatants passedthrough a 0.22 μm filter. The resulting phage lysates may be enumeratedand analyzed using the agar overlay technique. A similar number, and asimilar size of wild-type and φA1122::luxAB plaques may indicate thatintegration of luxAB into the φA1122 genome did not significantly impactthe ‘fitness’ of the phage.

Example 7 Alternative Methods to Integrate luxAB into the Phage Genome

Although the ability to manipulate phage and express foreign genes iswell established, integration of luxAB in a non-essential locationwithin the phage genome may have an effect on the viability and fitnessof the phage. It may also be possible that the location of luxAB, theluxAB sequence, and the addition of a strong promoter (albeit with a 3′terminator) may negatively influence phage viability. In view of this,optional and/or alternative methods may be used to integrate luxAB intothe phage genome. For example, one option is to target the luxAB genesto a different non-essential (e.g., predicted) genomic location, e.g.,gp19.3. In another example, a promoter with mis-matches to the consensuspromoter may be used in order to reduce promoter strength (although thismay also reduce luxAB expression, bioluminescence and hence, detectionsensitivity). Third, the luxAB cassette without a specific promoter maybe used which may rely on endogenous read-through from phage promoters.In another example, an expression cassette lacking the TL17transcriptional terminators may be used since the terminators mayprevent phage promoter read-through into genes located 3′ of the luxABgenes.

Isolation of the recombinant phage may be by screening for plaques whichexhibit a bioluminescent phenotype (See for example, Example 12 andFIGS. 3-4). An alternative method for the isolation of the recombinantphage may use a positive antibiotic selection pressure. For example, astreptomycin resistance gene may be included within the luxAB cassetteto provide a positive selection for the isolation of ‘streptomycinresistant phage’. To circumvent the concomitant use ofstreptomycin-tagged phage and the pathogen Y. pestis, an excision system(e.g., the CRE-loxP system of bacteriophage PI, the flp/frt system ofSaccharomyces cerevisiae, the Gin system of phage Mu, Pin system of E.coli prophage E14, etc.) may be used to excise the streptomycin genefollowing isolation of the recombinant phage. Such genetic tools ensurecompliance with the strict regulatory rules that are in place for thegenetic manipulation of bacterial pathogens.

Example 8 Analysis of the Ability of φA1122::luxAB to Detect Y. pestis

The ability of φA1122::luxAB to quickly, and sensitively detect Y.pestis may be required for a detection system. The φA1122::luxAB may beable to detect Y. pestis in three hours or less, two hours and less, orone hour or less. In some embodiments, the detection may be in less thanone hour, less than forty minutes, less than thirty minutes, less thantwenty minutes, less than fifteen minutes, less than fourteen minutes,less than thirteen minutes, less than twelve minutes, less than elevenminutes, less than ten minutes, or less than five minutes. Phage genesmay be expressed within 2-15 minutes post-infection. In someembodiments, bioluminescent light may be detected in about or less than12 minutes of infection of an Yersinia microorganism by a φA1122::luxABor other recombinant luxAB phage (See for Example, Example, 12 and FIG.4). Sensitivity, dose response, and the signal response time may beanalyzed. Furthermore, since φA1122::luxAB may be used for the detectionof Y. pestis under diverse environmental conditions (in the field, inclinical settings etc.): (i) the phage may be stable for extendedstorage periods under standard conditions; (ii) the phage may remaininfective over a range of pH and temperatures, and (iii) theφA1122::luxAB infected cultures may produce a stable light signal over arange of temperatures.

The attenuated Y. pestis A1122 strain (BEI#NR-15) may be for initialanalysis under BSL2 conditions.

A. Signal response time: The time required to generate a signal response(bioluminescence) may be assessed (also see Example 12 and FIG. 4). Y.pestis A1122 may be grown in BHI media at 30° C. until the mid-stages ofexponential growth. Approximately 1×10⁸ CFU/mL Y. pestis cells (based onColony Forming Units after overnight growth at 30° C.) may be mixed withφA1122::luxAB at an MOI of 10 at 30° C. Sample processing may not berequired. Bioluminescence may be measured over time using the Bio-TekSynergy HT microplate luminometer in the presence of the substraten-decanal (0.7% decanal, ‘flash’ bioluminescence). Negative controls mayconsist of phage or cells only. A detectable light signal fromφA1122::luxAB-infected Y. pestis may be generated within 3 minutes,within 4 minutes, within 5 minutes, within 6 minutes, within 7 minutes,within 8 minutes, within 9 minutes, within 10 minutes, within 11minutes, within 12 minutes, within 13 minutes, within 14 minutes, within15 minutes or within 20 minutes. For example, bioluminescence may bedetected from luxABM13-infected E. coli after only 7.5 minutes. Also,gene products of the T7 phage (closely related to the φA1122) may beexpressed extremely quickly at 2-8 minutes post-infection at 30° C. Seeresults in Example 12, FIG. 4 that demonstrate that φA1122::luxAB caneffectively and rapidly detect the presence of Y. pestis within 12minutes or lesser.

B. Detection of viable bacteria: The ability to detect only viable andpotentially infectious cells may be extremely beneficial and may be adistinct advantage over antigen and PCR tests which may detect thepresence of Y. pestis, but yield no information as to its infectivity.To demonstrate that φA1122::luxAB has the ability to detect viable cellsonly, cells may be killed by heat treatment at 65° C. for 10 min. Toensure the cells are not viable, the cells may be plated onto BHI agarand grown at 30° C. for 48 h; no colonies are expected to form.Heat-killed cells may be mixed with φA1122::luxAB and assayed forbioluminescence. A bioluminescent signal is not expected since phageinfection and light production may be dependent on active cellmetabolism (for phage replication, and luxAB expression). The resultsmay confirm that viable and/or potentially infectious cells may bedetected by φA1122::luxAB.

C. Assay sensitivity and dose response: The ability to detect lowconcentrations of cells and to relatively quantify the number of cellspresent may be important characteristics of a detection methodology.

To investigate assay sensitivity and dose-dependent characteristics, aten-fold serial dilution of cells ranging from 1×10⁸ to 1×10¹ CFU/mL maybe incubated with 1×10⁹ φA1122::luxAB plaque forming units (PFU) asdescribed above. Since an MOI of at least 10 may be used (MOI increasesas the cells number decreases), every cell may be infected. The resultsin Example 12 and FIG. 4 show the detection of bioluminescence within 12minutes upon infection of Yersinia pestis cells (at 1.03×10⁷ CFU/mL),cells (n=3), with phage (50 μl of 5×10¹⁰ PFU/mL stock), at amultiplicity of infection of approximately 10.

The results may demonstrate that as the number of cells decrease,bioluminescence decreases proportionally indicating dose-responsecharacteristics. The lowest number of detectable cells may bedetermined. As the number of cells decreases, the signal response timemay increase (takes longer to detect the bioluminescent signal due tofewer cells transduced and expressing luxAB).

The sensitivity of the assay may be for example, from about 500 CFU/mLto about 1,000 CFU/mL. Sensitivity of the assay may depend on theefficiency of infection, expression of the luxAB expression cassette,and the sensitivity of the luminometer. Since the human infectiousaerosolized dose of Y. pestis are estimated to be 100-500 CFU, thesensitivity of the φA1122::luxAB assay may be similar to the humaninfectious dose.

Example 9 LuxAB Expression and Stability at Different Temperatures

LuxAB proteins may be unstable at temperatures above 30° C. Althoughcell growth and bioluminescence assays may be performed at 30° C., LuxABthermostability may vary depending on the host species. For example, T7phages may infect and replicate faster at 37° C. than 30° C. Mature T7phage particles may be detected at 9 or 18 min after infection dependingwhether the incubation temperature is 30° C. or 37° C., respectively.Incubation at elevated temperatures may result in faster signal responsetimes. LuxAB thermostability may be analyzed in Yersinia independentlyfrom infection. Y. pestis may be grown in BHI media at 30° C.Exponentially growing cells (˜1×10⁸ CFU/mL) may be infected withφA1122::luxAB (˜5×10⁸ PFU/mL). The culture may be incubated at 30° C.for 10 min to allow phage absorption to the cells, and then dividedequally and incubated at various temperatures (10° C., 15° C., 20° C.,25° C., 30° C., and 35° C.). Bioluminescence may be monitored everyminute for 1-50 min. Since temperature changes may also influence ratesof luxAB expression (e.g., transcription and translation), changes inbioluminescence may not be strictly correlated with LuxAB stability.Crude protein lysates may then be prepared from φA1122::luxAB-infectedY. pestis and assayed for bioluminescence at different temperatures.Analysis of the crude protein lysates ensures that only preformed LuxABmay be detected and may provide an indication of LuxAB thermostabilityin Y. pestis.

Example 10 Phage Stability and Viability

An important aspect of whether the φA1122::luxAB phage may be suitablefor Y. pestis detection may depend on the stability of lysates afterlong-term storage and ability of the phage to remain infective under thediverse conditions that may be encountered (e.g., outside thelaboratory). Ideally, the phage may be resistant to changes in: (i) pHvalues; (ii) temperature, and (iii) light exposure. In general, phageare extremely stable and may survive a range of pH's (pH 4-10) andtemperatures (up to 60° C.). Moreover, phage may be freeze-dried for theproduction of field-able detection kits. Although long-term preservationmay be empirically determined for a specific phage, followinglyophilization phage may be stored (e.g., at room temperature or withcooling) without a decrease in titer for years if not indefinitely.

Prior to determining phage stability, φA1122::luxAB may be concentratedand purified using polyethylene glycol (PEG). 0.75M NaCl may be added tothe phage lysates and mixed continuously at 4° C. for 1 h to dissociatethe phage from the bacterial debris and media components. 10% PEG 8000may be added gradually, and the phage may be allowed to precipitate at4° C. overnight. The precipitated phage may be collected bycentrifugation (11,000×g, 15 min, 4° C.) and resuspended gently in SMbuffer.

To determine the stability of φA1122::luxAB at different pH's, the pH ofSM buffer may be adjusted to the following values using 1 M NaOH or 1 MHCl: pH 4, 6, 8, and 10. The purified φA1122::luxAB suspension (˜1×10¹⁰PFU/mL) may be diluted 1/200 into pH-adjusted SM buffer and stored atambient temperature or at 4° C. Both ambient and cold temperatures maybe tested since stability at different pH's is influenced by differentstorage temperatures. After 24 h incubation at the designatedtemperatures, the number of phage may be tittered using the agar overlaytechnique and compared to the number of viable phage in the originalstarting sample. φA1122::luxAB may remain viable over a range of pHvalues.

Since φA1122::luxAB may be used outside of the lab after months (if notyears) of storage, it may be desirable for phage to remain viable under‘standard’ conditions. To determine the stability of phage preparationsunder different storage conditions, purified phage lysates may be storedin SM buffer in the dark at 4° C., room temperature (approx. 19° C.),and 37° C. for different durations. Phage aliquots (100 μl) may beenumerated for plaques using the agar overlay technique after 1, 2, and3 months (and longer if possible) and compared to the original titer.

Example 11 Use of the Methods of the Disclosure to Detect Other Yersiniasp. and Use of Other Bacteriophage

Wild-type φA1122 phage has an extraordinary ability to infect most Y.pestis strains, and has been used by the CDC and the WHO for theconfirmed identification of Y. pestis. It has been shown that only twoY. pestis strains out of 1000's tested in the CDC collection have beenidentified as φA1122 resistant. A phage may infect most Y. pestisstrains due to the lack of diversity among Y. pestis strains which maybe due to the lack of opportunities to grow, infect, and evolve comparedto other bacterial species. A potential caveat of the phage detectionsystem, however, is the potential to infect the closely related speciesY. pseudotuberculosis. Although temperature may be used to differentiatethe species since the phage does not grow on Y. pseudotuberculosis at20° C., this may not be practical, especially for use outside of thelaboratory. To circumvent this one may identify (e.g., by microarray andin silico DNA analysis), and fuse a Y. pestis specific promoter toluxAB. Therefore, although φA1122::luxAB may infect Y.pseudotuberculosis, the luxAB promoter may not be expressed and lightmay not be produced. Alternatively, a cocktail of luxAB-Y. pestis phagemay be used, each phage tagged with a different version of theluciferase with which emits light at a different emission spectrum. Forexample, the recently sequenced L-413C Y. pestis phage (complete genomelisting GenBank Accession No: AY251033 and NC_(—)004745), has a verybroad host range within the species but in contrast to φA1122, is unableto infect Y. pseudotuberculosis.

Example 12 Isolation, Analysis and Detection of RecombinantφA1122::luxAB Phage

A. Isolation of Recombinant φA1122::luxAB Phage:

In one example, the luxAB cassette was targeted for integration into theYersinia φA1122 phage genome upstream of gene 0.3 by homologousrecombination. The φA1122 phage sequence at positions 897 to 903 wasreplaced with the luxAB cassette to generate a recombinant reporterphage with a genome size of 39,666 bp. φA1122::luxAB phage was isolatedby PCR screening of serially diluted phage until an individualrecombinant clone was isolated (FIG. 3).

B. Analysis of Recombinant φA1122::luxAB Phage:

To analyze whether the luxAB integration into φA1122 had occurred at thecorrect site in the phage genome, PCR primers were designed to span boththe 5′- and 3′-integration junction sites; each primer set was designedto ensure that primer binding occurred both within and without theoriginal integration cassette. For example, for the 5′- and3′-integration sites, primers were designed to bind either within therecombination cassette (luxAB) or in the phage φA1122 genome at either5′ or 3′ of the cassette. The predicted size of PCR products for the5′-junction, 3′-junction, and luxA were 591, 521, and 163 bp,respectively.

PCR analysis using the primers targeting the 5′ and 3′ integrationjunction sites generated PCR products of the correct predicted size(FIG. 3), indicating that the luxAB cassette integrated at the correctloci. The gel following the PCR analysis is depicted in FIG. 3 whereinin lane 1 has the PCR product, formed with the primers described above,in the absence of template (no product seen as expected for thiscontrol); lane 2 has the PCR product, formed with the primers describedabove, with the wild-type φA1122 phage (no product seen as expected forthis control); lane 3 has PCR product, formed with the primers describedabove, with the recombinant φA1122::luxAB phage (See products for the5′-junction, 3′-junction, and luxA at the predicted sizes of 591, 521,and 163 bp in lane 3). Thus, PCR analysis shows the presence of luxA andintegration of the luxAB into the φA1122 genome at the expectedlocation. The lane “M” in the gel in FIG. 3 has the molecular weightmarker that is a 100 bp marker DNA ladder.

PCR primers targeting luxA confirmed the presence of the reporter genes(FIG. 3). Since DNase 1-treated cell free phage supernatants were alsoable to transduce a bioluminescent phenotype to Y. pestis A1122, theseresults collectively indicated that functional φA1122::luxAB phage weregenerated. Titers of the recombinant phage were in the range of10¹⁰-10¹¹ plaque forming units/mL (PFU/mL). This titer is comparable totiters achievable with the wild-type φA1122 and suggests that thefitness of the recombinant phage was not compromised.

C. φA1122::luxAB Detection of Yersinia pestis

The ability of φA1122::luxAB to transduce a bioluminescent phenotype toY. pestis strain A1122 was assessed. Exponentially growing Y. pestis(OD₆₀₀ of approximately 0.2) were harvested and mixed with phage. Forexample, Y. pestis was grown in Luria Bertani medium at 28° C. withshaking at 225 rpm. At an OD600 of 0.185 (1.03×10⁷ CFU/mL), cells (n=3)were mixed with phage (50 μl of 5×10¹⁰ PFU/mL stock, a multiplicity ofinfection of approximately 10), and incubated at 28° C. with shaking at225 rpm. The ability of φA1122::luxAB to transduce bioluminescence(Relative Light Units, RLUs) was monitored over time using a Synergy IImultiplate detection reader, following the addition of a luciferasesubstrate such as 2% n-decanal (as depicted in FIG. 4).

A steady increase in bioluminescence was detected from Y. pestisphage-infected cells (FIG. 4). A detectable light signal abovebackground (phage alone or cells alone) was evident within 12 minutesafter phage infection. The results indicate that: (i) the φA1122::luxABphage were able to infect and transduce a bioluminescent phenotype to Y.pestis; (ii) the luxAB genes were functional in Y. pestis and produced asteady detectable bioluminescent signal, and (iii) a rapid signalresponse time at about 12 minutes after phage infection (FIG. 4, Seearrow). Controls consisted of cells or phage alone. Numbers are theaverage±SD of 3 infections (FIG. 4).

These equivalents and alternatives along with obvious changes andmodifications are intended to be included within the scope of thepresent disclosure. Moreover, one of ordinary skill in the art willappreciate that no embodiment, use, and/or advantage is intended touniversally control or exclude other embodiments, uses, and/oradvantages. Expressions of certainty (e.g., “will,” “are,” and “cannot”)may refer to one or a few example embodiments without necessarilyreferring to all embodiments of the disclosure. Accordingly, theforegoing disclosure is intended to be illustrative, but not limiting,of the scope of the disclosure.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Abremski, et al., 1986, J Biol Chem 261:391-6.-   Bossi, et al., 2006, Cell Mol Life Sci 63:2196-212.-   Calendar, et al., 2005, Oxford University Press.-   Carlson, 2005, CRC Press, Boca Raton.-   Choi, K. H., et al., 2008, Appl Environ Microbiol 74:1064-75.-   Conchas, R. F., et al., 1990, Gene 87:133-7.-   D'Aoust, J. Y., et al., 1988, J Dairy Sci 71:3230-6.-   Dennis, D. T., et al., 1999, Plague Manual: Epidemiology,    Distribution, Surveillance, and Control.-   Escher, A., et al., 1989, Proc Natl Acad Sci USA, 86:6528-32.-   Garcia, E., et al., 2003, J Bacteriol, 185:5248-62-   Garcia, E., et al., 2008, Virology, 372:85-96.-   Lin, L. Y., et al., 2004, Biochemistry, 43:3183-94.-   Liu, Q., et al., 1993, Proc Natl Acad Sci USA, 90:1761-5.-   Loessner, M. J., et al., 1996, Appl Environ Microbiol., 62:1133-40-   Mackey, B. M., et al., 1994, J Appl Bacteriol., 77:149-54.-   Meyer, K. F. 1974, Plague immunization. Journal of Infectious    Diseases 129:S13-S18.-   Sambrook, J., et al., 1989, Cold Spring Harbor Press, New York.-   Schofield, D. A., et al., 2001, J Bacteriol., 183:6947-50.-   Schofield, D. A., et al., 2002, Curr Microbiol., 44:425-30.-   Schofield, D. A., et al., 2002, FEMS Microbiol Lett., 215:237-42.-   Schofield, D. A., et al., 2003, Appl Environ Microbiol., 69:3385-92.-   Sternberg, N., et al., 1981, J Mol Biol., 150:467-86.-   Studier, F. W. 1981. J Mol Biol., 153:493-502.-   Westwater, C., et al., 2005, CRC Press, Boca Raton.-   Wright, J. J., et al., 1992, Embo J 11:1957-64.-   Young, R. 1992, Microbiol Rev., 56:430-81.-   Zierdt, C. H. 1988, Appl Environ Microbiol., 54:2590.

1. A Yersinia detection system comprising: a phage operable to infect aYersinia microorganism, the phage comprising a luxAB reporter nucleicacid configured and arranged to be expressed upon infection of theYersinia microorganism by the phage; and a detector operable to detectexpression of the luxAB reporter nucleic acid.
 2. The detection systemof claim 1, wherein the luxAB reporter nucleic acid is operably linkedto one or more Yersinia expression control elements.
 3. The detectionsystem of claim 2, wherein the one or more Yersinia expression controlelements are selected from the group consisting of transcriptionalcontrol elements, translational control elements and combinationsthereof.
 4. The detection system of claim 1, wherein the Yersiniamicroorganism is Yersinia pestis.
 5. The detection system of claim 1,wherein the Yersinia microorganism is selected from the group consistingof Yersinia enterocolitica, Yersinia pseudotuberculosis, andcombinations thereof.
 6. The detection system of claim 1, wherein thephage comprises a phage selected from the group consisting of serovar 1,serovar 2, serovar 3, and serovar
 4. 7. The detection system of claim 6,wherein the phage is a lytic phage.
 8. The detection system of claim 7,wherein the phage comprises a φA1122.
 9. The detection system of claim6, wherein the phage is a temperate phage.
 10. The detection system ofclaim 9, wherein the phage comprises a L-413C.
 11. A phage operable toinfect a Yersinia microorganism comprising a detectable reporterconfigured and arranged to be expressed in the Yersinia microorganism,the detectable reporter comprising a nucleic acid encoding a luxAB gene,and wherein the expression of the luxAB gene is detected asbioluminescent light.
 12. The phage of claim 11, wherein the detectablereporter further comprises at least one expression control elementoperably linked to the luxAB gene.
 13. The phage of claim 11, whereinthe phage comprises a phage selected from the group consisting ofserovar 1, serovar 2, serovar 3, and serovar
 4. 14. The phage of claim11, wherein the phage is a lytic phage.
 15. The phage of claim 14,wherein the phage comprises a φA1122.
 16. The phage of claim 11, whereinthe phage is a temperate phage.
 17. The phage of claim 16, wherein thephage comprises a L-413C.
 18. A method of detecting the presence of aYersinia microorganism in a test sample comprising: a) providing a phageoperable to infect a Yersinia microorganism, the phage comprising areporter configured and arranged to be expressed upon infection of theYersinia microorganism by the phage; b) contacting the test sample withthe phage under conditions that permits the phage to infect the Yersiniamicroorganism and express the reporter; and c) detecting expression ofthe reporter, wherein detecting the reporter indicates that the Yersiniamicroorganism is present in the test sample. 19-26. (canceled)
 27. Themethod of claim 18, wherein the reporter comprises a luxAB gene.
 28. Themethod of claim 27, wherein detecting the expression of the reportercomprises detecting bioluminescence.
 29. A method of claim 28, whereindetecting bioluminescence further comprises providing a substratespecific to the luxAB gene product.
 30. The method of claim 29, whereinthe substrate comprises an aldehyde. 31-32. (canceled)
 33. A kitcomprising: a) a phage operable to infect a Yersinia microorganism,comprising a reporter configured and arranged to be expressed uponinfection of the Yersinia microorganism by the phage, in a suitablecontainer; and b) one or more containers to mix the phage with a testsample that may comprise the Yersinia microorganism. 34-40. (canceled)